Optimised methods (SDS/PAGE and LC‐MS) reveal deamidation in all examined transglutaminase‐mediated reactions

Transglutaminases (TGs) are a family of structurally and functionally related enzymes that catalyse calcium‐dependent post‐translational modifications of proteins through protein–protein crosslinking, amine incorporation, or deamidation. For many years deamidation mediated by TGs was considered to be a side reaction, but recently substrate‐specific deamidations have been reported. Here we describe an optimised SDS/PAGE assay for the easy and rapid monitoring of the TG reaction with small peptides. The relative proportion of deamidation to transamidation was evaluated by densitometric analysis and confirmed by nano‐liquid chromatography–nano‐electrospray ionisation MS. We further investigated the effect of reaction conditions on transamidation and deamidation of TG1, TG2 and blood coagulation factor XIII A‐subunit (FXIII‐A) enzymes using a panel of glutamine‐containing peptide substrates. The ratio of transamidation to deamidation was enhanced at high excess of the acyl‐acceptor substrate and increasing pH. In addition, it was influenced by peptide substrates as well. Whereas deamidation was favoured at low cadaverine concentrations and acidic pH, no significant effect of calcium was observed on the ratio of transamidation/deamidation. Under our experimental conditions, deamidation always occurred in vitro even at high excess of the acyl‐acceptor substrate, and the reaction outcome was shifted to deamidation at neutral pH. Our results provide clear evidence of the deamidation in the TG reaction, and may serve as an important approach for in vivo analysis of deamidation to better understand the role of TGs in biological events.

Transglutaminases (TGs) are a family of structurally and functionally related enzymes that catalyse calcium-dependent post-translational modifications of proteins through protein-protein crosslinking, amine incorporation, or deamidation. For many years deamidation mediated by TGs was considered to be a side reaction, but recently substrate-specific deamidations have been reported. Here we describe an optimised SDS/PAGE assay for the easy and rapid monitoring of the TG reaction with small peptides. The relative proportion of deamidation to transamidation was evaluated by densitometric analysis and confirmed by nano-liquid chromatographynano-electrospray ionisation MS. We further investigated the effect of reaction conditions on transamidation and deamidation of TG1, TG2 and blood coagulation factor XIII A-subunit (FXIII-A) enzymes using a panel of glutamine-containing peptide substrates. The ratio of transamidation to deamidation was enhanced at high excess of the acyl-acceptor substrate and increasing pH. In addition, it was influenced by peptide substrates as well. Whereas deamidation was favoured at low cadaverine concentrations and acidic pH, no significant effect of calcium was observed on the ratio of transamidation/deamidation. Under our experimental conditions, deamidation always occurred in vitro even at high excess of the acyl-acceptor substrate, and the reaction outcome was shifted to deamidation at neutral pH. Our results provide clear evidence of the deamidation in the TG reaction, and may serve as an important approach for in vivo analysis of deamidation to better understand the role of TGs in biological events.
The distribution and physiological roles of TGs have been investigated in various cell types and tissues. TG2 is the most studied member of the TGs family. This multifunctional protein has diverse cellular localisation and is implicated in several physiological (regulation of cell survival/death processes, cell adhesion, migration, signal transduction, proliferation) and pathological processes (coeliac disease, neurodegenerative disorders, fibrosis, inflammatory diseases, metabolic diseases and cancer) [2]. TG1 mainly exists in the upper spinous and granular layers of the skin; it is involved in the terminal differentiation of keratinocytes by the formation of the crosslinked cell envelope [3]. Thrombin-activated factor FXIII-A plays an essential role in the stabilisation of fibrin clots and in wound healing through the formation of isopeptide bonds [4].
TGs catalyse Ca 2+ -dependent post-translational modifications of proteins by generating a protein-protein crosslink (between a specific c-carboxamide group of a glutamine and an e-amino group of a lysine side chain), amine incorporation or deamidation. In the first step active site cysteine reacts with the c-glutaminyl group of the proteins or peptides leading to the formation of a thioester intermediate. In the second step the acyl group is transferred to an amine substrate resulting in the formation of an isopeptide bond or the water can act as an alternative nucleophile leading to site-specific deamidation of the glutamine residue [5,6]. Many of these crosslinking reactions occur within 5-10 min, for example in case of FXIII-A between glutamine (Gln398 or -399) and lysine (Lys406) residues of the fibrin c-chains [7], whereas crosslinking of fibrin a-chains involving Gln221, -237, -328, or -366 and Lys208 or -606 takes place more slower [4,8].
Deamidation of glutamine and asparagine residues is one of the most prevalent post-translational modifications and converts an uncharged amino acid to a negatively charged residue introducing alternations in the protein's conformation. The protein function can be changed as it is determined by its global structure, and electrostatic protein-protein interaction can be modulated as well [9]. Deamidation can occur in different ways (non-enzymatically, by glutamines or phosphateactivated glutaminases) regulating several biological processes [10-12]. Moreover, cytotoxic necrotising factors in Escherichia coli and necrotoxin in Bordetella, which are considered to be functional relatives of TGs, can induce the formation of stress fibres by the deamidation of Rho proteins [13].
Deamidation by TGs was believed to be a side reaction, taking place only in the absence of primary amines or at low pH when availability of amines is limited [5], but recently it has been reported that selective deamidation in small heat shock protein [14] and bB2and bB3crystallins [15] can occur in a substrate-specific manner. Respectively, the substrate affinity and reaction conditions can influence the propensity for deamidation and transamidation [16]. Many research works are focusing on the examination of TG crosslinking activity, which is connected to some diseases such as fibrosis and neurodegeneration. So far TG-mediated deamidation activity has been related to coeliac disease [17,18], and only a few reports have been published on its role in other physiopathological processes [15, [19][20][21]. These studies support the potential role of TG-dependent deamidation in the regulation of biological processes.
In this study we report an optimised SDS/PAGE assay for the rapid and easy detection of both transamidated and deamidated peptides. TG1, TG2, FXIII-A enzymes and a panel of glutamine-containing peptides were examined to determine how the ratio of deamidation to transamidation can be influenced by some reaction parameters, such as affinity for substrates, amine donor concentration, Ca 2+ concentration and pH. The identity and the relative quantity of the reaction products were confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis. Our results are well correlated with the published data, cited above. Unexpectedly, under our reaction conditions, we found that a particular deamidation always occurs in vitro, even at high excess of the acyl-acceptor substrates.

Synthetic peptide substrates
The peptides and their derivatives [fluorescein amidite (FAM)-labelled peptides and deaminated controls] were synthesis by Covalab according to the established method for the production of synthetic peptides using solid-phase peptide synthesis described by Merrifield et al. [22]. Their purity was determined by analytical and preparative reversed-phase HPLC and mass spectrometry. All peptides were dissolved in DMSO at a final concentration of 10 mM. Isoenzyme-specific glutamine-containing peptides have been reported by Sugimura et al. [23,24] and used to develop specific TG activity assays [25,26]. K9 is a natural TG2 sequence based on b-casein [27]. Among the natural reactive glutamine site chains in fibrinogen a-chains a short peptide sequence containing Q238 [8] was chosen for our experiments. The corresponding deamidated control peptides were synthesised by Covalab (Table 1).

Transglutaminase reaction
Five micromolar of specific glutamine donor substrates labelled with fluorescent FAM and cadaverine (5-1000 lM) as an acyl acceptor were incubated with their corresponding isoenzymes at 37°C; 18 mUÁmL À1 of TG1, TG2, or thrombin (1 UÁmL À1 )-activated FXIII-A was applied in the assay buffer (20 mM Tris pH 7.2 or 8.0 or MES pH 5.0 or 6.0, 150 mM NaCl, 10 mM DTT) in the presence or absence of 10 mM EDTA. pH was 7.2, unless the effect of pH (5-8) was examined. The enzymatic reaction was initiated by the addition of 5 mM CaCl 2 , except in the experiments in which the effect of Ca 2+ was investigated (0.1-5 mM).

SDS/PAGE assay
The assay was previously reported by Kenniston et al. [28] and modified as briefly described below. After the incubation period the enzymatic reaction was stopped by boiling the sample in 69 SDS-loading buffer (9.3 w/w DTT, 12 wt% SDS, 47 v/v% glycerol, 0.06 wt% bromophenol blue in 0.5 M Tris/HCl, pH 6.8). The reaction products were run on SDS/PAGE (15% T, 2.6% C; T represents the total concentration of polyacrylamide monomer expressed in g per 100 mL and C is the percentage of bis-polyacrylamide) and visualised by fluorescence detection (Luminescent Image Analyzer LAS-1000 plus; Fujifilm, Dusseldorf, Germany).

Liquid chromatography-mass spectrometry analysis
The samples were diluted 10 times in a solution of 0.1% formic acid before analysis. Mass spectrometry analysis was performed on a linear ion trap LTQ Velos (Thermo Scientific, San Jose, CA, USA) with nano-electrospray ionisation (ESI) source coupled in-line to a nanoRSLC system Ultimate 3000 (Thermo Scientific, Germering, Germany). One microlitre of sample was injected via the autosampler. Samples were first desalted and concentrated on a reverse phase precolumn (C18 PepMap100, 300 lm i.d. 9 5 mm, 5 lm, 100 A; Thermo Scientific) for 3 min at 20 lLÁmin À1 with H 2 O/acetonitrile 98/2-0.1% formic acid. Samples were then separated on a nanocolumn (Acclaim C18, 15 cm 9 75 lm i.d, 2 lm; Thermo Scientific). The HPLC gradient was 5-55% solvent B (A = 5% acetonitrile, 0.1% formic acid; B = 80% acetonitrile, 0.1% formic acid) for 30 min followed by 5 min 99% B. The total duration was set to 50 min at a flow rate of 300 nLÁmin À1 . The oven temperature was kept constant at 40°C.
MS spectra were recorded in the mass range m/z 500-1100 in positive ionisation mode; the enhanced scan rate was used for the full MS spectrum.

Data analysis
Relative intensity of fluorescent bands was analysed with IMAGEJ and plotted with PRISM 5 (GraphPad Software Inc., San Diego, CA, USA) software.

Determination of transamidation and deamidation rates by transglutaminases
Post-translational modification (PTM) is one of the powerful regulatory elements that confer a specific function to each protein. Dysregulation of PTMs has been the object of a number of studies and was shown to be associated with several diseases [29]. Among the different type of PTMs, deamidation is unique as it has been shown to occur in different ways: spontaneously, chemically and enzymatically [30].
Deamidation is the conversion of selected glutamine and also asparagine residues into glutamate and aspartate/isoaspartate through hydrolytic reaction. Such reaction requires only water, is conditioned by both sequence and structure, and is facilitated by physicochemical conditions such as high temperatures, extreme pH or high ionic strength.
Deamidation of specific proteins such as adrenocorticotropin [31] and lens crystallins [32] has been shown to have biological repercussions due to folding changes and/or modification of life-span. Although some proteins can undergo the deamidation reaction with little or no loss of biological activity, others do lose activity, which can be associated with a growing list of pathological phenomena such as age-related, neurological and autoimmune diseases [33].
While in vivo deamidation of Asn to Asp was clearly established, TG-mediated deamidation of Gln is not fully understood and rather it was believed to be only a side-reaction, occurring when the second substrate is not available or at acidic pH. However, over about a decade many reports focused on the study of the mechanism of hydrolysis of the specific Gln residues in order to elucidate the role of TGs in the physiopathology of some diseases and coeliac disease in particular. In order to evaluate the deamidation reaction of TG to convert Gln to Glu, several methods were used depending on whether the Gln substrates were natural (e.g. proteins) or synthetic (e.g. peptides). In the case of proteins, deamidations were analysed through molecular modelling and biological activity [34][35][36] whereas synthetic peptides were analysed by various techniques such as mass spectrometry (MS and LC-MS), size exclusion chromatography, capillary electrophoresis, HPLC, 2D gels and western blot [14].
In this work we optimised an electrophoresis assay based on SDS/PAGE for the rapid and easy detection of both transamidated and deamidated peptides. All reaction products of FAM fluorescein-labelled peptides could be simultaneously detected with high resolution and specificity. The relative ratio of deamidation and transamidation was evaluated by densitometric analysis (Fig. 1B,E). The identity and the proportion of the reaction products were confirmed by nano-LC-nano-ESI-MS analysis. The relative quantities were calculated from extraction ions chromatogram areas of the doubly charged ions of each species (Fig. 1C,F, Figs S1, S2, Table S1), assuming that all the species have the same response factors. The data obtained by LC-MS are in good correlation with the results of densitometric analysis, indicating that the optimised SDS/ PAGE assay is a reliable method for the semi-quantitative examination of both transamidated and deamidated reaction products.

Catalytic activity of transglutaminases always generates deamidated peptide products
In this study TG1, TG2, FXIII-A enzymes and five glutamine-containing peptides (K5, T26, F11, K9 and aC(Q328)) ( Table 1) were examined to determine how the ratio of deamidation to transamidation can be influenced by some reaction parameters such as substrates affinity, amine donor concentration, Ca 2+ concentration and pH. K5 [23], T26 and F11 [24] were described as isoenzymes preferring glutamine substrates having high affinity to TG1, TG2 and FXIII-A, respectively, whereas K9 [27] and fibrinogen aC(Q328) peptides [8] are known as natural glutamine donor substrates for TG2 and FXIII-A. Applying high excess of the amine donor substrate (cadaverine), the ratio of transamidation to deamidation was increased, whereas deamidation was favoured at low cadaverine concentrations. In the absence of the second substrate, no crosslinked product was detected (Fig. 2). In addition this effect appeared to be substrate dependent and confirm the results obtained by the group of Sollid [16]. TG2 transamidase activity was rather higher with K9 (Fig. 2B) than with T26 peptide (Fig. 2C) indicating better recognition of natural glutamine substrate. This difference was not observed with FXIII-A as the reactivity is higher with F11 than with fibrinogen aC(Q328) peptides. Indeed upon the same reaction conditions the relative ratio of deamidated/transamidated fibrinogen aC(Q328) peptide was increased (Fig. 2E) whereas F11 peptide was preferred for transamidation (Fig. 2D). This slight difference in the deamidation/transamidation rate may be explained by the influence of the neighbouring amino acids relative to the targeted Gln and may affect the affinity of the enzymes. Indeed Boros et al. [15] reported that TG2 can process site-specific deamidation, and a proline at position +2 to the specific glutamine residue may positively influence deamidation [35].
Moreover, the results reveal that TG2-and FXIII-A-catalysed substrate deamidation could always occur as an excess of primary amine did not completely inhibit deamidation (Fig. 1B-E). However, deamidation by TG1 is less obvious in all conditions studied and this could explain why K5 may not be good substrate of this enzyme for deamidation ( Fig. 2A). Using natural and good substrate for TG1 will be of interest in completing this work.

Effect of pH on the transglutaminase-catalysed deamidation
Fleckenstein et al. [35] described that the TG2-catalysed reaction of gliadin substrate is strongly influenced by pH. Here, we extended the investigation for two other isoenzymes and five peptide substrates as detailed in the Materials and methods (Fig. 3). In all cases the transamidation and deamidation reactions were analysed in buffers with pH values ranging from 5 to 8. TG1 (Fig. 3A) and FXIII-A (Fig. 3D,E) seem to not be active at acidic pH, but at pH 6.0 fibrinogen aC(Q328) peptide was deamidated in the majority of cases (Fig. 3E). At pH 8.0 all the peptides were converted to transamidated forms by all the corresponding enzymes. In the condition of neutral pH, TG1 was not active due probably to the low kinetic reaction, whereas TG2 and FXIII-A were able to convert the peptides to transamidated and deamidated products. The proportion of each product depends on the type of the enzyme and also the glutamine substrate. T26 was highly deamidated by TG2 whereas the natural substrate K9 was in the majority of cases transamidated (Fig. 3B,C) and with FXIII-A the deamidation was more important than the transamidation for both substrates (Fig. 3D,E). These results further demonstrate that the rate of the transamidation reactions is significantly increased at alkaline pH indicating the importance of the nucleophily of the amine, which must be unprotonated. As the pKa of the cadaverine amine is around 10, it is expected that the transamidation reaction can be favoured over deamidation. Indeed in the report of Fleckenstein et al. [35] using 5-(biotinamido)penthylamine as an amine donor with pK a around 10.5, a general base-catalysed deacylation mechanism was proposed for the transamidation reaction through a nucleophilic attack on the thiol ester intermediate. Increasing the pH to narrow the pK a of the base would decrease its protonation, and consequently the competition by water molecules is blocked explaining the increase of transamidation rate.

Effect of Ca 2+ on the transglutaminase-catalysed deamidation
It is well known that Ca 2+ is required for the activation of TGs through inducing a large conformational change in the enzyme structure [37,38]. Because of the large difference in the open and closed conformations, we hypothesised that depending on Ca 2+ binding, more transient conformers with a different hydrodynamic radius can be exhibited. Probably at lower Ca 2+ concentrations the substrate-binding channel could be particularly covered and the entrance of the second substrate could be inhibited resulting on the deamidation of the glutamine donor substrate via hydrolysis. Transamidation and deamidation were analysed with different Ca 2+ concentrations ranging from 0.1 to 5 mM as described in Materials and methods (Fig. 4). At lower Ca 2+ concentration (0.1-0.2 mM) TG2 seems to not be active (Fig. 4B,C) whereas TG1 converted the majority of K5 to transamidated product (Fig. 4A). With FXIII-A we observed high activity with F11 substrate (Fig. 4D) and low activity with natural substrate (Fig. 4E), which can be explained by the high affinity of the enzyme to the synthetic substrate. By increasing Ca 2+ concentration the enzymes were more active and both transamidation and deamidation occurred at a different rate depending on the Gln substrate: transamidation was observed more with TG1 and TG2 using K5 and T26 substrates, respectively, and more deamidation was obtained with TG2 and FXIII-A using K9 and fibrinogen aC(Q328). Based on these results no significant effect of calcium on the ratio of transamidation/deamidation was obtained.
In conclusion, our assay based on SDS/PAGE provides an easy and rapid method for monitoring the TG reaction with synthetic small peptides. The  fractions of native, deamidated and transamidated peptides can be successfully separated and their rates were confirmed by nano-LC-nano-ESI-MS. Under our experimental conditions we noticed that a particular deamidation always occurs, even at high molar concentration (148-200-fold molar excess) of amine donor substrate and it is preferred at low to neutral pH. These data provide further evidence for the simultaneous reactions of transamidation and deamidation, but further work will be needed to confirm this in vitro and in vivo at the endogenous proteins level. Knowledge of the in vivo regulation of transamidation versus deamidation is therefore crucial for the elucidation of the TG enigma. Most of the TG activity assays are based on detection of the transamidated products. Since the deamidation is no longer believed to be a side reaction, TG activity cannot be accurately evaluated only by detecting the crosslinked products.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.   Table S1. Molecular mass and m/z (mass-to-charge ratio) of K9 and fibrinogen aC(325-336) glutamine donor peptides.